The field of the invention is diagnosis of and therapy for leukemia.
Leukemias including, but not limited to, acute leukemias such as acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are among the most common malignancies in children. Myelodysplastic syndrome is a designation for a group of syndromes similar to preleukemia (see, e.g. The Merck Manual, 16th ed., Berkow et al., Eds., Merck Research Laboratories, Rahway, N.J., pp. 1243-1245). Leukemias are also a serious cause of morbidity and mortality among adult humans, although MLL gene translocations are present in perhaps only a small proportion of adult acute leukemias. The incidences of ALL and AML in the United States are, respectively, 20 and 10.6 per million individuals per year in infants less than one year old. The aggressiveness with which a leukemia is treated depends, in part, on whether the leukemia has as its genesis a rearrangement of a portion of a chromosome at one or more particular sites. Some translocations may be detected by karyotype analysis, and others cannot be detected by such analysis.
Translocation of the MLL gene (which is alternately designated ALL-1, Htrx1, or HRX) at chromosome band 11q23 is associated with most cases of ALL which occur during infancy and with most monoblastic variants of AML which occur during the first four years of life (Cimino et al., 1993, Blood 82:544-546; Pui et al., 1995, Leukemia 9:762-769; Hilden et al., 1995, Blood 86:3876-3882; Chen et al., 1993, Blood 81:2386-2393; Martinez-Climent et al., 1993, Leukemia 9:1299-1304). About five percent of de novo cases of adult acute leukemia and most DNA topoisomerase II inhibitor-related leukemias are associated with similar translocations (Pui et al., 1995, supra; Martinez-Climent et al., 1993, supra; Raimondi, 1993, Blood 81:2237-2251; Felix et al., 1995, supra).
The MLL gene is 90 kilobases long, comprises 36 exons, and encodes a 3969 amino acid residue protein (Rasio et al., 1996, Cancer Res. 56:1766-1769). The MLL gene is believed to be involved in hematopoiesis and leukemogenesis. The MLL gene product contains several structural motifs important in the regulation of transcription (Domer et al., 1993, Proc. Natl. Acad. Sci. USA 90:7884-7888; Djabali et al., 1992, Nature Genet. 2:113-118; Gu et al., 1992, Cell 71:701-708; Tkachuk et al., 1992, Cell 71:691-700; Ma et al., 1993, Proc. Natl. Acad. Sci. USA 90:6350-6354) and functions as a positive regulator of Hox gene expression (Yu et al., 1995, Nature 378:505-508). Translocation of the MLL gene at chromosome band 11q23 disrupts an 8.3 kilobase breakpoint cluster region (ber) which is interposed between exons 5 and 11 of MLL. Approximately thirty different translocation partner genes of MLL have been recognized (Martinez-Climent et al., 1993, supra; Raimondi, Blood 81:2237-2251; Felix et al., 1995, Blood 85:3250-3256). Many of these partner genes have not been cloned or characterized.
MLL gene translocations may be detected by karyotype analysis as terminal 11q23 deletions (Shannon et al., 1993, Genes Chromosomes Cancer 7:204-208; Prasad et al., 1993, Cancer Res. 53:5624-5628; Yamamoto et al., 1994, Blood 83:2912-2921). About one third of ALL cases are associated with MLL rearrangements that cannot be detected by karyotype analysis. (Sorenson et al., 1992, Blood 80:255a; Schichman et al., 1994, Proc. Natl. Acad. Sci. USA 91:6236-6239; Schichman et al., 1994, Cancer Res. 54:4277-4280).
Sites of chromosome rearrangement (hereinafter, xe2x80x9cbreakpoint regionsxe2x80x9d) have been localized to introns within the bcr of MLL in several de novo cases of leukemia (Gu et al., 1992, Proc. Natl. Acad. Sci. USA 89:10464-10468; Negrini et al., 1993, Cancer Res. 53:4489-4492; Domer et al., 1993, Proc. Natl. Acad. Sci. USA 90:7884-7888; Corral et al., 1993, Proc. Natl. Acad. Sci. USA 90:8538-8542; Gu et al., 1994, Cancer Res. 54:2327-2330). The location of breakpoint regions within MLL and the identity of the nucleotide sequences located at such breakpoint regions are believed to vary according to etiology and pathogenesis of the leukemia. Fewer than half of the about thirty known MLL translocation partner genes have been cloned and identified, although for many of these partner genes, only partial or cDNA sequences are known.
One determinant of the location of a breakpoint region may be the nucleotide sequence preference attributable to either DNA topoisomerase II or a complex comprising DNA topoisomerase II and an agent which interacts with DNA topoisomerase II (Liu et al., 1991, In: DNA Topoisomerases in Cancer, Oxford University Press, New York, pp. 13-22; Ross et al., 1988, In: Important Advances in Oncology, pp.65-79; Pommier et al., 1991, Nucl. Acids Res. 19:5973-5980; Pommier, 1993, Cancer Chemother. Pharmacol. 32:103-108). For example, epipodophyllotoxins form a complex with DNA and DNA topoisomerase II, whereby chromosomal breakage can be effected at the site of complex formation (Corbett et al., 1993, Chem. Res. Toxicol. 6:585-597). Epipodophyllotoxins and other DNA topoisomerase II inhibitors have been associated with leukemias characterized by heterogenous translocations throughout the bcr of MLL at chromosome band 11q23 (Pui et al., 1991, N. Engl. J. Med. 325:1682-1687; Pui et al., 1990 Lancet 336:417-421; Winick et al., J. Clin. Oncol. 11:209-217; Broeker et al., 1996, Blood 87:1912-1922; Felix et al., 1993, Cancer Res. 53:2954-2956; Felix et al., 1995, Blood, 85:3250-3256; Pedersen-Bjergaard, 1992, Leukemia Res. 16:61-65; Pedersen-Bjergaard, 1991, Blood 78:1147-1148).
DNA topoisomerase II catalyzes transient double-strand breakage and religation of genomic DNA, and is involved in regulating DNA topology by relaxation of supercoiled genomic DNA. It is believed that agents which interact with DNA topoisomerase II and which are associated with leukemias inhibit the ability of DNA topoisomerase II to catalyze religation following double-strand breakage. One suggested model for translocations involving MLL entails DNA topoisomerase II-mediated chromosome breakage within the bcr, followed by fusion of DNA free ends from different chromosomes mediated by cellular DNA repair mechanisms (Felix et al., 1995, Cancer Res. 55:4287-4292). Although not strictly inhibitors in the enzymatic sense, epipodophyllotoxins are designated DNA topoisomerase II inhibitors because they decrease the rate of chromosomal religation catalyzed by DNA topoisomerase II and stabilize the DNA topoisomerase II-DNA covalent intermediate (Chen et al., 1994, Annu. Rev. Pharmacol. Toxicol. 84:191-218; Osheroff, 1989, Biochemistry 28:6157-6160; Chen et al., 1984, J. Biol. Chem. 259:13560-13566; Wang et al., 1990, Cell 62:403-406; Long et al., 1985, Cancer Res. 45:3106-3112; Epstein, 1988, Lancet 1:521-524; Osheroff et al., 1991, In: DNA Topoisomerases in Cancer, Potmesil et al., Eds., Oxford University Press, New York, pp. 230-239).
Chromatin structure and scaffold attachment regions may also affect the location of a breakpoint within bcr (Broeker et al., 1996, Blood 87:1912-1922).
Abasic sites are produced by oxidative DNA damage, ionizing radiation, alkylating agents, and spontaneous DNA hydrolysis (Kingma et al., 1995, J. Biol. Chem. 270:21441-21444). Abasic sites are the most common form of spontaneous DNA damage. Abasic sites resulting from exposure to environmental toxins or spontaneous abasic sites may be important mediators of leukemogenesis and provide another explanation of how chromosomal breakage is initiated in leukemia in infants (Kingma et al., 1997, Biochemistry 36:5934-5939), because abasic sites increase DNA topoisomerase II-mediated breakage.
Panhandle PCR methods have been described, and can be used to amplify genomic DNA having a nucleotide sequence comprising a known sequence which flanks an unknown sequence located 3xe2x80x2 with respect to the known sequence (Jones et al., 1993, PCR Meth. Applicat. 2:197-203; U.S. Pat. No. 5,411,875). The panhandle PCR methods comprise generation of a single-stranded DNA having a sequence comprising a region of known sequence at the 5xe2x80x2-end of the single-stranded DNA followed by a region of unknown sequence and having a region complementary to known region DNA at the 3xe2x80x2-end of the single-stranded DNA. The complementary region is complementary to a portion of DNA within the region of known sequence. Thus, the template comprises regions at each end having known sequences. Using primers complementary to each of these regions, the section of the template comprising region of unknown sequence may be amplified, and the nucleotide sequence of this section may be determined. Panhandle PCR has not been used to identify translocation breakpoints or to clone translocation partner genes.
There remains a need for a method of identifying and characterizing MLL rearrangement in individual patients afflicted with leukemia. Identification and characterization of such a rearrangement in the genome of a patient indicates the type and aggressiveness of therapy which may be provided to the patient to treat the leukemia and symptoms associated therewith. The present invention provides such a method.
The invention includes a method of amplifying an unknown region which flanks a known region of a leukemia-associated DNA sequence. The method comprises (a) providing a template polynucleotide comprising a sense strand which comprises the known region and the unknown region, wherein the unknown region is nearer the 3xe2x80x2-end of the sense strand than is the known region, wherein the known region comprises a first portion and a second portion, and wherein the first portion is nearer the unknown region than is the second portion; (b) ligating a loop-forming oligonucleotide to the 3xe2x80x2-end of the sense strand, wherein the loop-forming oligonucleotide is complementary to the first portion; (c) annealing the loop-forming oligonucleotide with the first portion to generate a panhandle structure; (d) subjecting the panhandle structure to extension, whereby an additional region complementary to the second portion is generated at the free end of the loop-forming oligonucleotide; and (e) subjecting the panhandle structure to PCR in the presence of a first primer homologous with the second portion, whereby the unknown region is amplified.
In one aspect, the leukemia-associated DNA sequence comprises MLL.
In a preferred embodiment, the known region comprises a portion of the breakpoint cluster region of MLL.
In another preferred embodiment, the known region comprises a portion of an exon of MLL selected from the group consisting of exon 5 and exon 11.
In another aspect, the loop-forming oligonucleotide has a nucleotide sequence comprising SEQ ID NO: 4.
In yet another aspect, the first primer has a nucleotide sequence selected from the group consisting of SEQ ID NO: 5-8.
In yet a further aspect, the panhandle structure is subjected to PCR in the presence of the first primer and further in the presence of a second primer, wherein the second primer is nested with respect to the first primer.
In a preferred embodiment, each of the first primer and the second primer independently has a nucleotide sequence selected from the group consisting of SEQ ID NO: 5-8.
In another aspect, the template polynucleotide further comprises an antisense strand, wherein the 5xe2x80x2-end of the antisense strand overhangs the 3xe2x80x2-end of the sense strand, and wherein a portion of the loop-forming oligonucleotide is complementary to the overhanging region of the antisense strand.
In yet another aspect, the template polynucleotide is provided by obtaining genomic DNA from a patient; contacting the genomic DNA with a restriction endonuclease, whereby a genomic DNA fragment is generated, the genomic DNA fragment comprising the known region and the unknown region, whereby the genomic DNA is the template polynucleotide.
The invention also includes a variant method of amplifying an unknown region which flanks a known region of a leukemia-associated DNA sequence. This method comprises (a) providing a template polynucleotide comprising an antisense strand which comprises a region complementary to the known region and a region complementary to the unknown region, wherein the region complementary to the unknown region is nearer the 5xe2x80x2-end of the antisense strand than is the region complementary to the known region, wherein the known region comprises a first portion and a second portion, and wherein the first portion is nearer the unknown region than is the second portion; (b) ligating a first oligonucleotide to the 5xe2x80x2-end of the antisense strand, wherein the first oligonucleotide is homologous with the first portion; (c) annealing a pre-template polynucleotide with the antisense strand, the pre-template polynucleotide being homologous with the second portion; (d) subjecting the pre-template polynucleotide to extension, whereby a sense strand is generated, the sense strand comprising the known region, the unknown region, and a loop-forming oligonucleotide at the 3xe2x80x2-end thereof, the loop-forming oligonucleotide being complementary to the first portion; (e) annealing the loop-forming oligonucleotide with the first portion to generate a panhandle structure; (f) subjecting the panhandle structure to extension, whereby an additional region complementary to the second portion is generated at the free end of the loop-forming oligonucleotide; and (g) subjecting the panhandle structure to PCR in the presence of a first primer homologous with the second portion, whereby the unknown region is amplified.
In one aspect of this aspect of the invention, prior to ligating the first oligonucleotide to the antisense strand, a bridging oligonucleotide is annealed with a portion of the antisense strand adjacent the 5xe2x80x2-end thereof and the first oligonucleotide is annealed with the bridging oligonucleotide.
Also included in the invention is a method of identifying a translocation partner of a leukemia-associated DNA sequence, the translocation partner comprising an unknown region, and the leukemia-associated DNA sequence comprising a known region. This method comprises (a) providing a template polynucleotide comprising a sense strand which comprises the known region and the unknown region, wherein the unknown region is nearer the 3xe2x80x2-end of the sense strand than is the known region, wherein the known region comprises a first portion and a second portion, and wherein the first portion is nearer the unknown region than is the second portion; (b) ligating a loop-forming oligonucleotide to the 3xe2x80x2-end of the sense strand, wherein the loop-forming oligonucleotide is complementary to the first portion; (c) annealing the loop-forming oligonucleotide with the first portion to generate a panhandle structure; (d) subjecting the panhandle structure to extension, whereby an additional region complementary to the second portion is generated at the free end of the loop-forming oligonucleotide; (e) subjecting the panhandle structure to PCR in the presence of a first primer homologous with the second portion, whereby the unknown region is amplified; and (f) identifying a portion of a human DNA sequence homologous with the unknown region, whereby the human DNA sequence is identified as the translocation partner.
The invention further includes a variant method of identifying a translocation partner of a leukemia-associated DNA sequence, the translocation partner comprising an unknown region, and the DNA sequence comprising a known region. This method comprises (a) providing a template polynucleotide comprising an antisense strand which comprises a region complementary to the known region and a region complementary to the unknown region, wherein the region complementary to the unknown region is nearer the 5xe2x80x2-end of the antisense strand than is the region complementary to the known region, wherein the known region comprises a first portion and a second portion, and wherein the first portion is nearer the unknown region than is the second portion; (b) ligating a first oligonucleotide to the 5xe2x80x2-end of the antisense strand, wherein the first oligonucleotide is homologous with the first portion; (c) annealing a pre-template polynucleotide with the antisense strand, the pre-template polynucleotide being homologous with the second portion; (d) subjecting the pre-template polynucleotide to extension, whereby a sense strand is generated, the sense strand comprising the known region, the unknown region, and a loop-forming oligonucleotide at the 3xe2x80x2-end thereof, the loop-forming oligonucleotide being complementary to the first portion; (e) annealing the loop-forming oligonucleotide with the first portion to generate a panhandle structure; (f) subjecting the panhandle structure to extension, whereby an additional region complementary to the second portion is generated at the free end of the loop-forming oligonucleotide; (g) subjecting the panhandle structure to PCR in the presence of a first primer homologous with the second portion, whereby the unknown region is amplified; and (h) identifying a portion of a human DNA sequence homologous with the unknown region, whereby the human DNA sequence is identified as the translocation partner.
Also included in the invention is a kit for panhandle PCR amplification of an unknown region of DNA which flanks a known region of the sense strand of a leukemia-associated DNA sequence. The kit comprises an oligonucleotide selected from the group consisting of an oligonucleotide which is complementary to the known region of the sense strand and an oligonucleotide which is homologous with the known region of the sense strand; and a first primer homologous with the known region of the sense strand.
In one aspect, the kit further comprises an internal primer, wherein the internal primer is nested with respect to the first primer, and wherein the internal primer is selected from the group consisting of a primer homologous with the known region of the sense strand.
In another aspect, the kit further comprises at least one recombination PCR primer.
In yet another aspect, the kit further comprises a restriction endonuclease; at least one reagent for ligating the oligonucleotide to a DNA strand obtained from a human patient; at least one reagent for extending a polynucleotide; and at least one reagent for performing PCR.
The invention also includes an isolated polynucleotide having a nucleotide sequence which comprises a sequence selected from the group consisting of SEQ ID NOs: 1-3.
In addition, the invention includes a primer derived from an isolated polynucleotide having a nucleotide sequence which comprises a sequence selected from the group consisting of SEQ ID NOs: 1-3.